Methods for detecting enhanced nmda receptor function and uses thereof

ABSTRACT

A method of determining the risk of cognitive decline in an aging subject is provided. The method includes analyzing an MRNA transcript including a GRIN2B nucleic acid sequence for the presence of the A allele in a biological sample obtained from the subject. The method also includes identifying the subject as having a decreased risk of cognitive decline when the A allele is present.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/589,257, filed May 8, 2017, which claims priority to U.S. ProvisionalPatent Application No. 62/341,883, which was filed in the U.S. Patentand Trademark Office on May 26, 2016, each of which are incorporatedherein by reference in its entirety for all purposes.

GOVERNMENTAL RIGHTS

This invention was made with government support under grants P30AG028383 and KO1 AG000986 awarded by NIH. The government has certainrights in the invention.

FIELD OF THE INVENTION

The disclosure provides methods for predicting memory performance anddetermining the risk for cognitive decline.

BACKGROUND OF THE INVENTION

The earliest and the most severe memory loss in old age and dementiaoccur in recent memory and working memory. Compared to young adults,older adults exhibit deficits in working memory, an online memorymechanism that allows manipulation of a current target image amongstdistracting stimuli. Remote memory or long-term is better retained.Evidence from neuropsychological and neuroimaging studies of visualworking memory indicates that working memory relies on activation of theventral temporal cortex, and top-down feedback from the prefrontal andmedial temporal cortex, and the hippocampus. It has been observed thatthere are significant individual differences in cognitive aging amonghealthy cognitively intact older adults due to genetic, learning andenvironmental factors. Since genetic factors influence working memoryperformance in the aging brain and the exact underlying mechanisms arenot well understood, there is a need in the art to identify geneticpolymorphisms critical to the molecular foundation of learning andmemory in individuals' brain processing speed and cortical responsesduring a working memory task.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1 depicts a schematic showing that the A allele of SNP r53764030creates an E twenty-six (ETS) transcription factor binding site in thepromoter region of the human GRIN2B (GRIN2B gene in italics) gene, whichencodes the GluN2B subunit of the N-methyl-D-aspartate (NMDA) receptor.

FIG. 2 depicts binding of recombinant Elk-1 protein (an ETStranscription factor) to double stranded DNAs containing the singlenucleotide polymorphism (SNP) r53764030 variant A allele from the humanGRIN2B gene promoter. Double-stranded DNA targets (Methods) weresequentially incubated with 1 μg recombinant human Elk-1 protein in EMSAbuffer, then incubated with the Elk-1 antibody 20 minutes. DNA-proteincomplexes were Samples were electrophoresed through 6% polyacrylamidegels in HEPES buffer pH 6.3, without polydldC. The gel was then stainedwith the SYBR Green to visualize DNA and SYPRO Ruby for protein bound toDNA. The stained gel was scanned using G:BOX (Syngene, US) for imaging.Elk-1 bound to DNA is indicated by the arrow.

FIG. 3 depicts a concentration dependent response of human GRIN2Bpromoter in N2a cells carrying either the A or G alleles of SNPr53764030 to NMDA. Luciferase reporter gene plasmids were constructed inpGL 4.10 (to produce Firefly luciferase, Promega, US). Luciferasereporter plasmids and the pGL4.75 plasmid (to produce Renilla luciferaseas a reference) were co-transfected into retinoic acid differentiatedN2a cells and assayed for (Methods). After 24 hours of transfection, thecells were incubated for six hours with 0, 30, 50, 70 or 90 μM NMDA fortranscription factors binding and then replaced with complete media(with 10% FBS) and cultured for an additional 40-44 hrs. Data arepresented as means ±SE.

FIG. 4A and FIG. 4B depict graphs showing the mean reaction time withincreasing age. Data was examined by analysis of covariance (ANCOVA),based on stratification by A allele carrier genotype group differences.Performance as mean reaction time versus age in GG homozygotes is shownin FIG. 4A, while mean reaction time versus age in A allele carriers (AAand AG genotypes combined) is shown in FIG. 4B. A significant differencein the slopes of the regression lines was observed (−3.994±SE 3.460 forA allele carriers vs 5.848±SE 2.281 for G/G; P=0.026).

FIG. 5A and 5B depict graphs showing the mean reaction time withincreasing age. Data was examined by analysis of covariance (ANCOVA),based on stratification by A allele carrier genotype group differences.Performance as mean reaction time versus age in GG homozygotes is shownin FIG. 5A, while mean reaction time versus age in A allele carriers (AAand AG genotypes combined) is shown in FIG. 5B. The slopes of theregression lines were significantly different. For A allele carriers asin FIG. 5B, the slope is −3.58±SE 0.21, versus GG genotype as in FIG. 5Ahaving a slope of 13.1±SE 0.20, t(8)=58.5, p<0.001).

DETAILED DESCRIPTION OF THE INVENTION

Described herein is the discovery that a G>A single nucleotidepolymorphism (SNP r53764030) that is 310 bps upstream of thetranscription start site for the human GRIN2B gene promoter creates anE-twenty six (ETS) transcription factor binding site. Accordingly, thisdisclosure is the first report that SNP r53764030, which results in theA allele, affects GRIN2B mRNA levels. An association of thisgain-of-function SNP in the GRIN2B gene and memory performance in anormally aged population of adults was demonstrated. Accordingly, SNPr53764030 within the promoter of the GRIN2B gene could be used topredict brain responses and memory performance during working memory.Further, it was shown that older subjects with the A allele performedbetter than those without the allele. This data suggests that enhancinglevels of GRIN2B protein during aging protects subjects from memory lossin later years. Additionally, predicting a subject's memory performanceduring aging by assessing for the presence of the A allele (SNPr53764030) allows for earlier intervention thereby slowing theprogression of aging effects on the brain.

Various aspects of the disclosure are described in more detail below.

I. Methods of Detection

In an aspect, the disclosure provides a method to classify a subjectbased on the presence of the GRIN2B A allele in a biological sampleobtained from the subject. The method generally comprises: (a) analyzingGRIN2B nucleic acid for the presence of the A allele; and (b)classifying the subject as having the A allele if GGAA is detected about310 base pairs upstream of the transcription start site for GRIN2B. Thesubject may be classified as having the A allele if they are eitherhomozygous for the A allele or heterozygous for the A allele. A subjecthomozygous for the A allele has only has the GGAA sequence about 310base pairs upstream of the transcription start site for GRIN2B, and maybe referred to as A/A. A subject heterozygous for the A allele has theGGAA sequence about 310 base pairs upstream of the transcription startsite for GRIN2B and a GGGA sequence about 310 base pairs upstream of thetranscription start site for GRIN2B, and may be referred to as A/G. Askilled artisan would be able to determine the transcription start sitefor GRIN2B. More specifically, a subject has the A allele if the SNPr53764030 is detected. SNP r53764030 is located on chromosome 12 atposition 13980398. The sequence information for r53764030 may be foundat ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=rs3764030. As used herein,“GRIN2B” refers to the human form of nucleic acid that encodes for theglutamate [NMDA] receptor subunit GluN2B or epsilon-2 protein. Theglutamate [NMDA] receptor subunit epsilon-2 protein may also be referredto as N-methyl D-aspartate receptor subtype 2B, GRIN2B, NMDAR2B, GluN2B,or NR2B. Other suitable terms for GRIN2B include EIEE27, GluN2B, MRD6,NMDAR2B, NR2B, and hNR3. As used herein, the “A allele” refers to asequence of GRIN2B comprising GGAA about 310 base pairs upstream of thetranscription start site for GRIN2B. The “A allele” also refers to SNPr53764030.

In another aspect, the disclosure provides a method of predicting memoryperformance in a subject. The method comprises: (a) analyzing GRIN2Bnucleic acid for the presence of the A allele in a biological sampleobtained from the subject; and (b) identifying the subject as havingimproved memory performance 1 or more years following analysis relativeto a reference value when the A allele is present. In an embodiment, thereference value is memory performance in a subject of the same agewithout the A allele. In another embodiment, the reference value is astandard value associated with the memory performance of a group ofsubjects of the same age without the A allele. The subject is predictedto have improved memory performance relative to a reference value 1 ormore years following the detection of the A allele. For example, thesubject is predicted to have improved memory performance relative to areference value about 1 year, about 2 years, about 3 years, about 4years, about 5 years, about 6 years, about 7 years, about 8 years, about9 years, about 10 years, about 11 years, about 12 years, about 13 years,about 14 years, about 15 years, about 16 years, about 17 years, about 18years, about 19 years, about 20 years, about 25 years, about 30 years,about 35 years, about 40 years, about 45 years, about 50 years, or morethan 50 years following the detection of the A allele. Memoryperformance may be measured by measuring sensory memory, short-termmemory (also referred to as working memory), and/or long-term memory.Long term memory includes explicit memories (conscious) and implicitmemories (unconscious). Explicit memory includes declarative memorywhich includes episodic memory and semantic memory. Implicit memoryincludes procedural memory. Any of the aforementioned types of memorymay be used to measure memory performance. In a specific embodiment,short-term memory (also known as working memory) may be used to measurememory performance. Methods of measuring memory performance are known inthe art. Non-limiting examples of methods of measuring memoryperformance include measuring reaction time, measuring accuracy ofbehavioral responses, and measuring recall. Numerous tests are known tomeasure memory including the reading span task, the operation spanktask, the rotation spank task, the verbal updating task, the numericalupdating task, the spatial-figural updating task, the recall 1-backtask, binding tasks, secondary memory tasks, and reasoning tasks.Specifically, the methods described in the Examples may be used tomeasure memory performance.

In still another aspect, the disclosure provides a method of predictingreaction time in a subject. The method comprises: (a) analyzing GRIN2Bnucleic acid for the presence of the A allele in a biological sampleobtained from the subject; and (b) identifying the subject as havingimproved reaction time 1 or more years following analysis relative to areference value when the A allele is present. In an embodiment, thereference value is reaction time in a subject of the same age withoutthe A allele. In another embodiment, the reference value is a standardvalue associated with the reaction time of a group of subjects of thesame age without the A allele. The subject is predicted to have improvedreaction time relative to a reference value 1 or more years followingthe detection of the A allele. For example, the subject is predicted tohave improved reaction time relative to a reference value about 1 year,about 2 years, about 3 years, about 4 years, about 5 years, about 6years, about 7 years, about 8 years, about 9 years, about 10 years,about 11 years, about 12 years, about 13 years, about 14 years, about 15years, about 16 years, about 17 years, about 18 years, about 19 years,about 20 years, about 25 years, about 30 years, about 35 years, about 40years, about 45 years, about 50 years, or more than 50 years followingthe detection of the A allele. Methods of measuring reaction time areknown in the art. Specifically, reaction time may be measured asdescribed in the Examples.

In still yet another aspect, the disclosure provides a method ofdetermining the risk of cognitive decline in an aging subject. Themethod comprises: (a) analyzing GRIN2B nucleic acid for the presence ofthe A allele in a biological sample obtained from the subject; and (b)identifying the subject as having a decreased risk of cognitive declinewhen the A allele is present. As used herein, “cognitive decline” refersto an impairment of cognition or memory that represents a deteriorationfrom a previous level of function. Non-limiting examples of measures ofcognitive decline include memory, reaction time, learning, thinking,language, judgment, decision-making, and motor coordination. Diseases ordisorders associated with cognitive decline include dementia,Alzheimer's disease, delirium, and amnesia. Accordingly, the disclosurealso provides a method of determining the risk of dementia in an agingsubject comprising: (a) analyzing GRIN2B nucleic acid for the presenceof the A allele in a biological sample obtained from the subject; and(b) identifying the subject as having a decreased risk of dementia whenthe A allele is present. As used herein, an “aging subject” is a subject30 years or more. For example, the subject may be 30 years or more, 35years or more, 40 years or more, 45 years or more, 50 years or more, 55years or more, 60 years or more, 65 years or more, 70 years or more, 75years or more, 80 years or more, 85 years or more, 90 years or more, 95years or more, or 100 years or more. In an embodiment, the methodfurther comprises treating a subject if the A allele is absent.Non-limiting examples of treatment include drugs to boostneurotransmitter levels (e.g., Aricept, Razadyne and Exelon), drugs toregulated the activity of the neurotransmitter glutamate (e.g.,memantine), occupational therapy, environmental approaches (e.g., reduceclutter and/or noise), donepezil, vitamin E, diet and exercise, andcognitive rehabilitation.

In other aspects, the disclosure provides a method of determiningtreatment of an aging subject. The method comprises: (a) analyzingGRIN2B nucleic acid for the presence of the A allele in a biologicalsample obtained from the subject; (b) classifying the subject as havingthe A allele if GGAA is detected about 310 base pairs upstream of thetranscription start site for GRIN2B or not having the A allele if GGGAis detected about 310 base pairs upstream of the transcription startsite for GRIN2B; and (c) treating the subject if the A allele is notdetected. The absence of the A allele may be indicative of cognitivedecline upon aging. For example, the absence of the A allele may beindicative of declines in memory upon aging. Further, the absence of theA allele may be indicative of dementia upon aging. Accordingly, if the Aallele is not detected, the subject may be more aggressively treatedrelative to a subject possessing the A allele. Non-limiting examples oftreatment for memory decline, cognitive decline and/or dementia includedrugs to boost neurotransmitter levels (e.g., Aricept, Razadyne andExelon), drugs to regulated the activity of the neurotransmitterglutamate (e.g., memantine), occupational therapy, environmentalapproaches (e.g., reduce clutter and/or noise), donepezil, vitamin E,diet and exercise, and cognitive rehabilitation.

In any of the foregoing embodiments, the subject may or may not bediagnosed with cognitive decline, memory loss and/or dementia. Incertain embodiments, the subject may not be diagnosed with cognitivedecline, memory loss and/or dementia but is suspected of havingcognitive decline, memory loss and/or dementia based on symptoms.Non-limiting examples of symptoms of cognitive decline, memory lossand/or dementia that may lead to a diagnosis include confusion, poormotor coordination, loss of short-term or long-term memory, identityconfusion, impaired judgment, emotional outbursts, isolation, and/ordulled or nonexistent emotions. In other embodiments, the subject maynot be diagnosed with cognitive decline, memory loss and/or dementia butis at risk of having cognitive decline, memory loss and/or dementia.Non-limiting examples of risk factors for cognitive decline, memory lossand/or dementia include family history, aging, cardiovascular disease,diabetes, smoking, depression, high blood pressure, elevatedcholesterol, lack of physical exercise, and infrequent participation inmentally or socially stimulating activities. In other embodiments, thesubject has no symptoms and/or no risk factors for cognitive decline,memory loss and/or dementia. Methods of diagnosing cognitive decline,memory loss and/or dementia are known in the art. Non-limiting examplesof method of diagnosing cognitive decline, memory loss and/or dementiainclude a thorough medical history, neurological exam to test reflexes,eye movements and/or walking and balance, blood tests, brain imaging,and/or mental status testing.

Suitable subjects include, but are not limited to, a human, a livestockanimal, a companion animal, a lab animal, and a zoological animal. Inone embodiment, the subject may be a rodent, e.g. a mouse, a rat, aguinea pig, etc. In another embodiment, the subject may be a livestockanimal. Non-limiting examples of suitable livestock animals may includepigs, cows, horses, goats, sheep, llamas and alpacas. In yet anotherembodiment, the subject may be a companion animal. Non-limiting examplesof companion animals may include pets such as dogs, cats, rabbits, andbirds. In yet another embodiment, the subject may be a zoologicalanimal. As used herein, a “zoological animal” refers to an animal thatmay be found in a zoo. Such animals may include non-human primates,large cats, wolves, and bears. In an embodiment, the animal is alaboratory animal. Non-limiting examples of a laboratory animal mayinclude rodents, canines, felines, and non-human primates. In certainembodiments, the animal is a rodent. In a preferred embodiment, thesubject is human.

The human subject may be of any age. However, since cognitive decline isnormally associated with aging, a human subject may be an older humansubject. In some embodiments, the human subject may be about 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 years of age or older. Insome preferred embodiments, the human subject is 30 years of age orolder. In other preferred embodiments, the human subject is 40 years ofage or older. In other preferred embodiments, the human subject is 45years of age or older. In yet other preferred embodiments, the humansubject is 50 years of age or older. In still other preferredembodiments, the human subject is 55 years of age or older. In otherpreferred embodiments, the human subject is 60 years of age or older. Inyet other preferred embodiments, the human subject is 65 years of age orolder. In still other preferred embodiments, the human subject is 70years of age or older. In other preferred embodiments, the human subjectis 75 years of age or older. In still other preferred embodiments, thehuman subject is 80 years of age or older. In yet other preferredembodiments, the human subject is 85 years of age or older. In stillother preferred embodiments, the human subject is 90 years of age orolder.

(a) Biological Sample

As used herein, the term “biological sample” refers to a sample obtainedfrom a human subject. Any biological sample containing GRIN2B nucleicacid is suitable. Numerous types of biological samples are known in theart. Suitable biological sample may include, but are not limited to,tissue samples or bodily fluids. In some embodiments, the biologicalsample is a tissue sample such as a tissue biopsy. The biopsied tissuemay be fixed, embedded in paraffin or plastic, and sectioned, or thebiopsied tissue may be frozen and cryosectioned. In other embodiments,the sample may be a bodily fluid. Non-limiting examples of suitablebodily fluids include blood, plasma, serum, peripheral blood, bonemarrow, urine, saliva, sputum, and cerebrospinal fluid. In a specificembodiment, the biological sample is blood, plasma, serum. In anotherspecific embodiment, the biological sample is blood. The fluid may beused “as is”, the cellular components may be isolated from the fluid, ora nucleic acid fraction may be isolated from the fluid using standardtechniques.

As will be appreciated by a skilled artisan, the method of collecting abiological sample can and will vary depending upon the nature of thebiological sample and the type of analysis to be performed. Any of avariety of methods generally known in the art may be utilized to collecta biological sample. Generally speaking, the method preferably maintainsthe integrity of the sample such that GRIN2B can be accurately detected.

(b) Analyzing GRIN2B Nucleic Acid

Typically, analyzing GRIN2B nucleic acid may comprise identifying thepresence of the A allele. As used herein, the “A allele” refers to asequence of GRIN2B comprising GGAA about 310 base pairs upstream of thetranscription start site for GRIN2B. The “A allele” also refers to SNPr53764030. The sequence surrounding SNP rs 3764030 comprises SEQ ID NO:1(CGCGTCAGTGTGCCCCTTCCAAGAAATGCCCAGTGTGCACCCCGTGCACAATCAGAACCCATTCAGCACAAGCCCGGGGTGGGAGGCGGCGCTGCTGCTGGAGGCGATGGGGAGAGCGAGCGAGACAAGTCAGCAGCAATGCAGATGGGGCTGGGGGCCGCACTCGCTGGCGAGTTAAGTGGGAATTGTGTTTCTGCGTGTGTGTGAGTGTGTGTTGCTGTATGGTGCCGCTTCTCCCCCCCTTCCTCCTTCCTTCCCACTTCCCTCCTTCGCTCGCTCCCTCCCTCTCTCCTCTTCCATTCAGGTTGGCTTTCCCACCTCTCATCCGTGCCTGTCCCAGGAATGGTATAGCCAGACCTTTTCTGAATTATTTATAGACCGGTACCAGCTGTTTTCAATTCCTCTCGTGTGCACTCTGTGGGAAATGCGGGGTTTCCTCCCCCCTTTCCTTAAAACGAATTGATATCTTTTTCGGAATGCATTTTTCTCACCCTCCGGGGRACACGCGAATCAAGCCCTGACCGCCTCTTTTTCCCCCTTAGGAAGGGGACGCTTTGGGAATGACCATGCTCCACCGAGGGACGGAGCCGGCCCCCAGCTTCTCCACACAGAGCCTCCTCCACTAACGCTCCAAAAACCAAAAACCGTAATTGCCAGAAGAAGCGTTAAAAATCTATTCCAGCCACTAACCTCACATGCACACGGAATAATTACTCTGGATTCTGCATTGTGAGCTGCTCTCCATACCCTGAATTACCTTTGAATTAAATCTTTTTTTTTTTGAATTTGCATCTCTTCAAGACACAAGATTAAAACAAAATTTACGCTAAATTGGATTTTAAATTATCTTCCGTTCATTTATCCTTCGTCTTTCTTATGTGGATATGCAAGCGAGAAGAAGGGACTGGACATTCCCAACATGCTCACTCCCTTAATCTGTCCGTCTAGAGGTTTGGCTTCTACAAACCAAGGTAGGGCAAATTCTATTTTATTTTTTCCTC), wherein R indicatesthe single nucleotide polymorphism where the nucleotide is either an Aor a G.

The A allele creates an E twenty-six (ETS) transcription factor bindingsite in the promoter region of the human GRIN2B nucleic acid. Thebinding of an ETS transcription factor increases the expression ofGRIN2B mRNA, synthesis of GRIN2B protein, or activity of GRIN2B proteinwhen compared to a sequence of GRIN2B gene without the A allele. Thepresence of the A allele may be identified using methods commonly knownin the art. Generally speaking, all or a portion of the promoter regionupstream of the GRIN2B nucleic acid sequence may be sequenced andcompared to SNP r53764030 (SEQ ID NO:1) to identify the A allele.

With the knowledge of the sequence of the A allele provided herein, itis a routine matter to design detection means such as primers and/orprobes that would be able to detect and/or identify the A allele.Possible techniques which might be utilized are well-established in theprior art and their use is readily adaptable by the skilled person forthe purposes of detecting the GRIN2B A allele disclosed herein. Forexample, amplification techniques may be used. Non-limiting examples ofamplification techniques may include polymerase chain reaction, ligasechain reaction, nucleic acid sequence based amplification (NASBA),strand displacement amplification (SDA), transcription mediatedamplification (TMA), Loop-Mediated Isothermal Amplification (LAMP),Q-beta replicase, Rolling circle amplification, 3SR, ramificationamplification (Zhang et al. (2001) Molecular Diagnosis 6 p141-150),multiplex ligation-dependent probe amplification (Schouten et al. (2002)Nucl. Ac. Res. 30 e57). Other related techniques for detecting mutationssuch as SNPs may include restriction fragment length polymorphism(RFLP), single strand conformation polymorphism (SSCP) and denaturinghigh performance liquid chromatography (DHPLC). A summary of many ofthese techniques can be found in “DNA Amplification: Currenttechnologies and applications” (Eds. Demidov & Broude (2004) Pub.Horizon Bioscience, ISBN:0-9545232-9-6) and other current textbooks.

Alternatively, probe based techniques may be used to detect the Aallele. For example, a nucleic acid probe that is complementary orhybridizable to the A allele may be used. The term “hybridize” or“hybridizable” refers to the sequence specific non-covalent bindinginteraction with a complementary nucleic acid. In a preferredembodiment, the hybridization is under high stringency conditions.Appropriate stringency conditions which promote hybridization are knownto those skilled in the art, or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. The term“probe” as used herein refers to a nucleic acid sequence that willhybridize to a nucleic acid target sequence (e.g. the A allele). Thelength of probe depends on the hybridization conditions and thesequences of the probe and nucleic acid target sequence. In oneembodiment, the probe is at least 8, 10, 15, 20, 25, 50, 75, 100, 150,200, 250, 400, 500 or more nucleotides in length. In some embodiments, aprobe comprises about 5, about 10, about 15, about 20, about 25, about30 bases upstream of the single nucleotide polymorphism (SNP) and about5, about 10, about 15, about 20 about 35, about 30 bases downstream ofthe SNP. For example, a probe may comprise the sequence set forth in SEQID NO:6 (CATCTCCGGGGAACACGCGAA). The probe may further comprise a labelfor detection of the presence of the A allele. Non-limiting examples ofsuitable labels include luminescent molecules, chemiluminescentmolecules, fluorochromes, fluorescent quenching agents, coloredmolecules, radioisotopes, scintillants, biotin, avidin, stretpavidin,protein A, protein G, antibodies or fragments thereof, polyhistidine,Ni2+, Flag tags, myc tags, heavy metals, and enzymes (including alkalinephosphatase, peroxidase, and luciferase).

(c) Determining the Risk of Cognitive Decline

A method of the disclosure comprises determining the risk of cognitivedecline. The level of risk is a measure of the probability of cognitivedecline occurring in a given individual. As used herein, “cognitivedecline” refers to an impairment of cognition or memory that representsa deterioration from a previous level of function. If the A allele isidentified, as described above, in a sample from a subject, then thesubject is at a lower risk (i.e., there is a decreased probability) ofdeveloping cognitive decline then a subject without the A allele. Forinstance, the risk may be less than about 5%, less than about 10%, lessthan about 15%, less than about 20%, less than about 25%, less thanabout 30%, less than about 35%, less than about 40%, less than about45%, or less than about 50%.

Alternatively, if the A allele is not identified in a sample from asubject, then the subject is at a higher risk of developing cognitivedecline. For instance, the risk may be greater than about 95%, greaterthan about 90%, greater than about 85%, greater than about 80%, greaterthan about 75%, greater than about 70%, greater than about 65%, greaterthan about 60%, greater than about 55%, or greater than about 50%.

Increased or decreased “risk” or “probability” may be determined, forexample, by comparison to the average risk or probability of anindividual subject developing cognitive decline within a definedpopulation. In this theoretical context, an increased risk for anindividual will mean that they are more than 50% likely to developcognitive decline within 5 years, whereas a reduced risk will mean thatthey are less than 50% likely to develop cognitive decline.

II. Methods of Treatment

In a different aspect, the disclosure provides a method to enhancememory in a subject. The method comprises administering a compositioncomprising a compound that increases GRIN2B activity or expression.Memory enhancement may be measured by measuring memory performance atone point in them and then measuring memory performance at a later pointin time, wherein when memory performance improves from the first pointin time to the second point in time, memory is enhanced. Memoryperformance may be measured by measuring sensory memory, short-termmemory (also referred to as working memory), and/or long-term memory.Long term memory includes explicit memories (conscious) and implicitmemories (unconscious). Explicit memory includes declarative memorywhich includes episodic memory and semantic memory. Implicit memoryincludes procedural memory. Any of the aforementioned types of memorymay be used to measure memory performance. In a specific embodiment,short-term memory (also known as working memory) may be used to measurememory performance. Methods of measuring memory performance are known inthe art. Non-limiting examples of methods of measuring memoryperformance include measuring reaction time, measuring accuracy ofbehavioral responses, and measuring recall. Numerous tests are known tomeasure memory including the reading span task, the operation spanktask, the rotation spank task, the verbal updating task, the numericalupdating task, the spatial-figural updating task, the recall 1-backtask, binding tasks, secondary memory tasks, and reasoning tasks.Specifically, the methods described in the Examples may be used tomeasure memory performance.

In another different aspect, the disclosure provides a method to protectagainst memory loss in a subject. The method comprises administering acomposition comprising a compound that increases GRIN2B activity orexpression. Measuring memory performance, as described above, may beused to measure memory loss.

In still another different aspect, the disclosure provides a method todelay the onset of cognitive decline in a subject. The method comprisesadministering a composition comprising a compound that increases GRIN2Bactivity or expression. As used herein, “cognitive decline” refers to animpairment of cognition or memory that represents a deterioration from aprevious level of function. Non-limiting examples of measures ofcognitive decline include memory, reaction time, learning, thinking,language, judgment, decision-making, and motor coordination. Diseases ordisorders associated with cognitive decline include dementia,Alzheimer's disease, delirium, and amnesia. Accordingly, the disclosurealso provides a method to delay the onset of dementia in a subjectcomprising administering a composition comprising a compound thatincreases GRIN2B activity or expression. For example, the onset ofcognitive decline may be delayed about 1 year, about 2 years, about 3years, about 4 years, about 5 years, about 6 years, about 7 years, about8 years, about 9 years, about 10 years, about 11 years, about 12 years,about 13 years, about 14 years, about 15 years, about 16 years, about 17years, about 18 years, about 19 years, about 20 years, about 25 years,about 30 years, about 35 years, about 40 years, about 45 years, about 50years, or more than 50 years following administration of a compositioncomprising a compound that increases GRIN2B activity or expression.

A subject may be as described above in Section I.

(a) Compositions

In an aspect, a composition comprises a compound that increases GRIN2Bactivity or expression. A composition may optionally comprise one ormore additional drug or therapeutically active agent in addition to acompound that increases GRIN2B activity or expression. A composition mayfurther comprise a pharmaceutically acceptable excipient, carrier ordiluent. Further, a composition may contain preserving agents,solubilizing agents, stabilizing agents, wetting agents, emulsifiers,sweeteners, colorants, odorants, salts (substances of the presentinvention may themselves be provided in the form of a pharmaceuticallyacceptable salt), buffers, coating agents or antioxidants.

Methods to determine if a compound increases GRIN2B activity orexpression are known in the art. For example, GRIN2B nucleic acidexpression, GRIN2B protein expression, or GRIN2B activity may bemeasured as described in more detail below.

A compound with the ability to increase GRIN2B activity or expressionmay potentially be used as a drug to prevent cognitive decline and/orimprove memory. A compound may include, without limitation, a drug, asmall molecule, a peptide, a nucleic acid molecule, a protein, anantibody, and combinations thereof. A nucleic acid molecule may be anantisense oligonucleotide, a ribozyme, a small nuclear RNA (snRNA), along noncoding RNA (LncRNA), or a nucleic acid molecule which formstriple helical structures. In certain embodiments, a compound with theability to increase GRIN2B activity or expression may be the GRIN2Bnucleic acid. For example, an expression vector encoding the GRIN2Bnucleic acid may be delivered to a cell using a viral vector or via anon-viral method of transfer. Viral vectors suitable for introducingnucleic acids into cells include retroviruses, adenoviruses,adeno-associated viruses, rhabdoviruses, and herpes viruses. Non-viralmethods of nucleic acid transfer include naked nucleic acid, liposomes,and protein/nucleic acid conjugates. An expression construct encodingthe GRIN2B nucleic acid that is introduced to the cell may be linear orcircular, may be single-stranded or double-stranded, and may be DNA,RNA, or any modification or combination thereof.

i. GRIN2B Nucleic Acid Expression

In an embodiment, GRIN2B nucleic acid expression may be measured toidentify a compound that increases GRIN2B. For example, when GRIN2Bnucleic acid expression is increased in the presence of a compoundrelative to an untreated control, the compound increased GRIN2B. In aspecific embodiment, GRIN2B mRNA may be measured to identify a compoundthat increased GRIN2B expression.

Methods for assessing an amount of nucleic acid expression in cells arewell known in the art, and all suitable methods for assessing an amountof nucleic acid expression known to one of skill in the art arecontemplated within the scope of the invention. The term “amount ofnucleic acid expression” or “level of nucleic acid expression” as usedherein refers to a measurable level of expression of the nucleic acids,such as, without limitation, the level of messenger RNA (mRNA)transcript expressed or a specific variant or other portion of the mRNA,the enzymatic or other activities of the nucleic acids, and the level ofa specific metabolite. The term “nucleic acid” includes DNA and RNA andcan be either double stranded or single stranded. Non-limiting examplesof suitable methods to assess an amount of nucleic acid expression mayinclude arrays, such as microarrays, polymerase chain reaction (PCR),such as RT-PCR (including quantitative RT-PCR), nuclease protectionassays and Northern blot analyses. In a specific embodiment, determiningthe amount of expression of a target nucleic acid comprises, in part,measuring the level of target nucleic acid mRNA expression.

In one embodiment, the amount of nucleic acid expression may bedetermined by using an array, such as a microarray. Methods of using anucleic acid microarray are well and widely known in the art. Forexample, a nucleic acid probe that is complementary or hybridizable toan expression product of a target gene may be used in the array. Theterm “hybridize” or “hybridizable” refers to the sequence specificnon-covalent binding interaction with a complementary nucleic acid. In apreferred embodiment, the hybridization is under high stringencyconditions. Appropriate stringency conditions which promotehybridization are known to those skilled in the art, or can be found inCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1 6.3.6. The term “probe” as used herein refers to a nucleic acidsequence that will hybridize to a nucleic acid target sequence. In oneexample, the probe hybridizes to an RNA product of the nucleic acid or anucleic acid sequence complementary thereof. The length of probe dependson the hybridization conditions and the sequences of the probe andnucleic acid target sequence. In one embodiment, the probe is at least8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500 or morenucleotides in length.

In another embodiment, the amount of nucleic acid expression may bedetermined using PCR. Methods of PCR are well and widely known in theart, and may include quantitative PCR, semi-quantitative PCR, multiplexPCR, or any combination thereof. Specifically, the amount of nucleicacid expression may be determined using quantitative RT-PCR. Methods ofperforming quantitative RT-PCR are common in the art. In such anembodiment, the primers used for quantitative RT-PCR may comprise aforward and reverse primer for a target gene. The term “primer” as usedherein refers to a nucleic acid sequence, whether occurring naturally asin a purified restriction digest or produced synthetically, which iscapable of acting as a point of synthesis when placed under conditionsin which synthesis of a primer extension product, which is complementaryto a nucleic acid strand is induced (e.g. in the presence of nucleotidesand an inducing agent such as DNA polymerase and at a suitabletemperature and pH). The primer must be sufficiently long to prime thesynthesis of the desired extension product in the presence of theinducing agent. The exact length of the primer will depend upon factors,including temperature, sequences of the primer and the methods used. Aprimer typically contains 15-25 or more nucleotides, although it cancontain less or more. The factors involved in determining theappropriate length of primer are readily known to one of ordinary skillin the art.

The amount of nucleic acid expression may be measured by measuring anentire mRNA transcript for a nucleic acid sequence, or measuring aportion of the mRNA transcript for a nucleic acid sequence. Forinstance, if a nucleic acid array is utilized to measure the amount ofmRNA expression, the array may comprise a probe for a portion of themRNA of the nucleic acid sequence of interest, or the array may comprisea probe for the full mRNA of the nucleic acid sequence of interest.Similarly, in a PCR reaction, the primers may be designed to amplify theentire cDNA sequence of the nucleic acid sequence of interest, or aportion of the cDNA sequence. One of skill in the art will recognizethat there is more than one set of primers that may be used to amplifyeither the entire cDNA or a portion of the cDNA for a nucleic acidsequence of interest. Methods of designing primers are known in the art.Methods of extracting RNA from a biological sample are known in the art.

The level of expression may or may not be normalized to the level of acontrol nucleic acid. Such a control nucleic acid should notspecifically hybridize with the GRIN2B nucleotide sequence. This allowscomparisons between assays that are performed on different occasions.

ii. GRIN Protein Expression

In another embodiment, GRIN2B protein expression may be measured toidentify a compound that increased GRIN2B expression. For example, whenGRIN2B protein expression is increased in the presence of a compoundrelative to an untreated control, the compound increased GRIN2B. In aspecific embodiment, GRIN2B protein expression may be measured usingimmunoblot.

Methods for assessing an amount of protein expression are well known inthe art, and all suitable methods for assessing an amount of proteinexpression known to one of skill in the art are contemplated within thescope of the invention. Non-limiting examples of suitable methods toassess an amount of protein expression may include epitope bindingagent-based methods and mass spectrometry based methods.

In some embodiments, the method to assess an amount of proteinexpression is mass spectrometry. By exploiting the intrinsic propertiesof mass and charge, mass spectrometry (MS) can resolve and confidentlyidentify a wide variety of complex compounds, including proteins.Traditional quantitative MS has used electrospray ionization (ESI)followed by tandem MS (MS/MS) (Chen et al., 2001; Zhong et al., 2001; Wuet al., 2000) while newer quantitative methods are being developed usingmatrix assisted laser desorption/ionization (MALDI) followed by time offlight (TOF) MS (Bucknall et al., 2002; Mirgorodskaya et al., 2000;Gobom et al., 2000). In accordance with the present invention, one canuse mass spectrometry to look for the level of protein encoded from atarget nucleic acid of the invention.

In some embodiments, the method to assess an amount of proteinexpression is an epitope binding agent-based method. As used herein, theterm “epitope binding agent” refers to an antibody, an aptamer, anucleic acid, an oligonucleic acid, an amino acid, a peptide, apolypeptide, a protein, a lipid, a metabolite, a small molecule, or afragment thereof that recognizes and is capable of binding to a targetgene protein. Nucleic acids may include RNA, DNA, and naturallyoccurring or synthetically created derivative.

As used herein, the term “antibody” generally means a polypeptide orprotein that recognizes and can bind to an epitope of an antigen. Anantibody, as used herein, may be a complete antibody as understood inthe art, i.e., consisting of two heavy chains and two light chains, ormay be any antibody-like molecule that has an antigen binding region,and includes, but is not limited to, antibody fragments such as Fab',Fab, F(ab')2, single domain antibodies, Fv, and single chain Fv. Theterm antibody also refers to a polyclonal antibody, a monoclonalantibody, a chimeric antibody and a humanized antibody. The techniquesfor preparing and using various antibody-based constructs and fragmentsare well known in the art. Means for preparing and characterizingantibodies are also well known in the art (See, e.g. Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, 1988; hereinincorporated by reference in its entirety).

As used herein, the term “aptamer” refers to a polynucleotide, generallya RNA or DNA that has a useful biological activity in terms ofbiochemical activity, molecular recognition or binding attributes.Usually, an aptamer has a molecular activity such as binging to a targetmolecule at a specific epitope (region). It is generally accepted thatan aptamer, which is specific in it binding to a polypeptide, may besynthesized and/or identified by in vitro evolution methods. Means forpreparing and characterizing aptamers, including by in vitro evolutionmethods, are well known in the art (See, e.g. U.S. Pat. No. 7,939,313;herein incorporated by reference in its entirety).

In general, an epitope binding agent-based method of assessing an amountof protein expression comprises contacting a sample comprising apolypeptide with an epitope binding agent specific for the polypeptideunder conditions effective to allow for formation of a complex betweenthe epitope binding agent and the polypeptide. Epitope bindingagent-based methods may occur in solution, or the epitope binding agentor sample may be immobilized on a solid surface. Non-limiting examplesof suitable surfaces include microtitre plates, test tubes, beads,resins, and other polymers.

An epitope binding agent may be attached to the substrate in a widevariety of ways, as will be appreciated by those in the art. The epitopebinding agent may either be synthesized first, with subsequentattachment to the substrate, or may be directly synthesized on thesubstrate. The substrate and the epitope binding agent may bederivatized with chemical functional groups for subsequent attachment ofthe two. For example, the substrate may be derivatized with a chemicalfunctional group including, but not limited to, amino groups, carboxylgroups, oxo groups or thiol groups. Using these functional groups, theepitope binding agent may be attached directly using the functionalgroups or indirectly using linkers.

The epitope binding agent may also be attached to the substratenon-covalently. For example, a biotinylated epitope binding agent may beprepared, which may bind to surfaces covalently coated withstreptavidin, resulting in attachment. Alternatively, an epitope bindingagent may be synthesized on the surface using techniques such asphotopolymerization and photolithography. Additional methods ofattaching epitope binding agents to solid surfaces and methods ofsynthesizing biomolecules on substrates are well known in the art, i.e.VLSIPS technology from Affymetrix (e.g., see U.S. Pat. No. 6,566,495,and Rockett and Dix, Xenobiotica 30(2): 155-177, both of which arehereby incorporated by reference in their entirety).

Contacting the sample with an epitope binding agent under effectiveconditions for a period of time sufficient to allow formation of acomplex generally involves adding the epitope binding agent compositionto the sample and incubating the mixture for a period of time longenough for the epitope binding agent to bind to any antigen present.After this time, the complex will be washed and the complex may bedetected by any method well known in the art. Methods of detecting theepitope binding agent-polypeptide complex are generally based on thedetection of a label or marker. The term “label”, as used herein, refersto any substance attached to an epitope binding agent, or othersubstrate material, in which the substance is detectable by a detectionmethod. Non-limiting examples of suitable labels include luminescentmolecules, chemiluminescent molecules, fluorochromes, fluorescentquenching agents, colored molecules, radioisotopes, scintillants,biotin, avidin, stretpavidin, protein A, protein G, antibodies orfragments thereof, polyhistidine, Ni2+, Flag tags, myc tags, heavymetals, and enzymes (including alkaline phosphatase, peroxidase, andluciferase). Methods of detecting an epitope binding agent-polypeptidecomplex based on the detection of a label or marker are well known inthe art.

In some embodiments, an epitope binding agent-based method is animmunoassay. Immunoassays can be run in a number of different formats.Generally speaking, immunoassays can be divided into two categories:competitive immmunoassays and non-competitive immunoassays. In acompetitive immunoassay, an unlabeled analyte in a sample competes withlabeled analyte to bind an antibody. Unbound analyte is washed away andthe bound analyte is measured. In a non-competitive immunoassay, theantibody is labeled, not the analyte. Non-competitive immunoassays mayuse one antibody (e.g. the capture antibody is labeled) or more than oneantibody (e.g. at least one capture antibody which is unlabeled and atleast one “capping” or detection antibody which is labeled.) Suitablelabels are described above.

In some embodiments, the epitope binding agent-based method is an ELISA.In other embodiments, the epitope binding agent-based method is aradioimmunoassay. In still other embodiments, the epitope bindingagent-based method is an immunoblot or Western blot. In alternativeembodiments, the epitope binding agent-based method is an array. Inanother embodiment, the epitope binding agent-based method is flowcytometry. In different embodiments, the epitope binding agent-basedmethod is immunohistochemistry (IHC). IHC uses an antibody to detect andquantify antigens in intact tissue samples. The tissue samples may befresh-frozen and/or formalin-fixed, paraffin-embedded (orplastic-embedded) tissue blocks prepared for study by IHC. Methods ofpreparing tissue block for study by IHC, as well as methods ofperforming IHC are well known in the art.

iii. GRIN2B Protein Activity

In an embodiment, GRIN2B activity may be measured to identify a compoundthat increases expression of GRIN2B. GRIN2B is involved in theefficiency of synaptic transmission. Accordingly, neuron firing may bemeasured as an indication of GRIN2B activity. Neuron signal transmissionmay be measured using methods standard in the art. For example, whenneuron signal transmission is increased in the presence of a compoundrelative to an untreated control, the compound increases GRIN2B.

In another embodiment, immunoblots may be performed against proteinsknown to interact with GRIN2B. Increased complexes of GRIN2B and theinteracting protein may indicate an increase in GRIN2B. Non-limitingexamples of protein known to interact with GRIN2B include actinin, alpha2, DLG2, DLG3, DLG4, EXOC4, KLOTHO, LIN7B, PSD-95, and RICS as well asother NMDA receptor subunits.

iv. Classification of DNA Sequence to Determine Efficacy For TreatmentUsing Composition and to Determine Candidates For Such Treatment

Compositions which act on NMDA receptors, for example by targeting theGRIN2B nucleic acid sequence, can be used to promote cognitive function.For example, memantine has been used as a drug for Alzheimer's disease.Memantine acts on NMDA receptors, targeting the GRIN2B subunit. Othermolecules that activate NMDA receptors, such as the known co-agonistsglycine and D-serine, can then activate GRIN2B gene expression. TheGRIN2B gene nucleic acid sequences can thus be used in predictingresponses of a drug. As such, the GRIN2B nucleic acid sequences can beused to determine whether a drug will be effective and, subsequently, todetermine candidates for treatment using the composition. By classifyingDNA sequences to determine whether the candidate has the A allele,comparative amounts of GRIN2B expression in a candidate individual canbe determined. The composition can then be used if applicable.

In at least one embodiment, the efficacy or response of a drug can bedetermined using in vitro methods. For example, cells can be screenedfor the drug or composition using GRIN2B nucleic acid sequences as aresponse. Presence of the A allele in the nucleic acid sequences fusedto a reporter gene such as an enzyme like lacZ (beta-galactosidase), ora fluorescent protein (green fluorescent protein or red fluorescentprotein) can be measured by but limited to histochemical staining,protein blots, liquid scintillation, spectrophotometry, luminometry, orchemiluminescence). In another example, cell viability using but limitedto assays that measure cytolysis (lactate dehydrogenase), dye exclusion(Trypan Blue), or mitochondrial activity by an endogenous oxidoreductaseenzyme that reduces a tetrazole to a formazan (MTT assay) that can bemeasured spectrophotometrically to determine drug response or efficacy.

v. Components of the Composition

The present disclosure also provides pharmaceutical compositions. Thepharmaceutical composition comprises a compound that increases GRIN2Bexpression or activity, as an active ingredient, and at least onepharmaceutically acceptable excipient.

The pharmaceutically acceptable excipient may be a diluent, a binder, afiller, a buffering agent, a pH modifying agent, a disintegrant, adispersant, a preservative, a lubricant, taste-masking agent, aflavoring agent, or a coloring agent. The amount and types of excipientsutilized to form pharmaceutical compositions may be selected accordingto known principles of pharmaceutical science.

In one embodiment, the excipient may be a diluent. The diluent may becompressible (i.e., plastically deformable) or abrasively brittle.Non-limiting examples of suitable compressible diluents includemicrocrystalline cellulose (MCC), cellulose derivatives, cellulosepowder, cellulose esters (i.e., acetate and butyrate mixed esters),ethyl cellulose, methyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, sodium carboxymethylcellulose, cornstarch, phosphated corn starch, pregelatinized corn starch, rice starch,potato starch, tapioca starch, starch-lactose, starch-calcium carbonate,sodium starch glycolate, glucose, fructose, lactose, lactosemonohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol,xylitol, maltodextrin, and trehalose. Non-limiting examples of suitableabrasively brittle diluents include dibasic calcium phosphate (anhydrousor dihydrate), calcium phosphate tribasic, calcium carbonate, andmagnesium carbonate.

In another embodiment, the excipient may be a binder. Suitable bindersinclude, but are not limited to, starches, pregelatinized starches,gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodiumcarboxymethylcellulose, ethylcellulose, polyacrylamides,polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fatty acid alcohol,polyethylene glycol, polyols, saccharides, oligosaccharides,polypeptides, oligopeptides, and combinations thereof.

In another embodiment, the excipient may be a filler. Suitable fillersinclude, but are not limited to, carbohydrates, inorganic compounds, andpolyvinylpyrrolidone. By way of non-limiting example, the filler may becalcium sulfate, both di- and tri-basic, starch, calcium carbonate,magnesium carbonate, microcrystalline cellulose, dibasic calciumphosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc,modified starches, lactose, sucrose, mannitol, or sorbitol.

In still another embodiment, the excipient may be a buffering agent.Representative examples of suitable buffering agents include, but arenot limited to, phosphates, carbonates, citrates, tris buffers, andbuffered saline salts (e.g., Tris buffered saline or phosphate bufferedsaline).

In various embodiments, the excipient may be a pH modifier. By way ofnon-limiting example, the pH modifying agent may be sodium carbonate,sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

In a further embodiment, the excipient may be a disintegrant. Thedisintegrant may be non-effervescent or effervescent. Suitable examplesof non-effervescent disintegrants include, but are not limited to,starches such as corn starch, potato starch, pregelatinized and modifiedstarches thereof, sweeteners, clays, such as bentonite,micro-crystalline cellulose, alginates, sodium starch glycolate, gumssuch as agar, guar, locust bean, karaya, pecitin, and tragacanth.Non-limiting examples of suitable effervescent disintegrants includesodium bicarbonate in combination with citric acid and sodiumbicarbonate in combination with tartaric acid.

In yet another embodiment, the excipient may be a dispersant ordispersing enhancing agent. Suitable dispersants may include, but arenot limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum,kaolin, bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose.

In another alternate embodiment, the excipient may be a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate,citric acid, sodium citrate; chelators such as EDTA or EGTA; andantimicrobials, such as parabens, chlorobutanol, or phenol.

In a further embodiment, the excipient may be a lubricant. Non-limitingexamples of suitable lubricants include minerals such as talc or silica;and fats such as vegetable stearin, magnesium stearate or stearic acid.

In yet another embodiment, the excipient may be a taste-masking agent.Taste-masking materials include cellulose ethers; polyethylene glycols;polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers;monoglycerides or triglycerides; acrylic polymers; mixtures of acrylicpolymers with cellulose ethers; cellulose acetate phthalate; andcombinations thereof.

In an alternate embodiment, the excipient may be a flavoring agent.Flavoring agents may be chosen from synthetic flavor oils and flavoringaromatics and/or natural oils, extracts from plants, leaves, flowers,fruits, and combinations thereof.

In still a further embodiment, the excipient may be a coloring agent.Suitable color additives include, but are not limited to, food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drugand cosmetic colors (Ext. D&C).

The weight fraction of the excipient or combination of excipients in thecomposition may be about 99% or less, about 97% or less, about 95% orless, about 90% or less, about 85% or less, about 80% or less, about 75%or less, about 70% or less, about 65% or less, about 60% or less, about55% or less, about 50% or less, about 45% or less, about 40% or less,about 35% or less, about 30% or less, about 25% or less, about 20% orless, about 15% or less, about 10% or less, about 5% or less, about 2%,or about 1% or less of the total weight of the composition.

The composition can be formulated into various dosage forms andadministered by a number of different means that will deliver atherapeutically effective amount of the active ingredient. Suchcompositions can be administered orally (e.g. inhalation), parenterally,or topically in dosage unit formulations containing conventionalnontoxic pharmaceutically acceptable carriers, adjuvants, and vehiclesas desired. Topical administration may also involve the use oftransdermal administration such as transdermal patches or iontophoresisdevices. The term parenteral as used herein includes subcutaneous,intravenous, intramuscular, intra-articular, or intrasternal injection,or infusion techniques. Formulation of drugs is discussed in, forexample, Gennaro, A. R., Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa. (18^(th) ed, 1995), and Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., NewYork, N.Y. (1980). In a specific embodiment, a composition may be a foodsupplement or a composition may be a cosmetic.

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, powders, pellets, and granules. In such solid dosageforms, the active ingredient is ordinarily combined with one or morepharmaceutically acceptable excipients, examples of which are detailedabove. Oral preparations may also be administered as aqueoussuspensions, elixirs, or syrups. For these, the active ingredient may becombined with various sweetening or flavoring agents, coloring agents,and, if so desired, emulsifying and/or suspending agents, as well asdiluents such as water, ethanol, glycerin, and combinations thereof. Foradministration by inhalation, the compounds are delivered in the form ofan aerosol spray from pressured container or dispenser which contains asuitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

For parenteral administration (including subcutaneous, intradermal,intravenous, intramuscular, intra-articular and intraperitoneal), thepreparation may be an aqueous or an oil-based solution. Aqueoussolutions may include a sterile diluent such as water, saline solution,a pharmaceutically acceptable polyol such as glycerol, propylene glycol,or other synthetic solvents; an antibacterial and/or antifungal agentsuch as benzyl alcohol, methyl paraben, chlorobutanol, phenol,thimerosal, and the like; an antioxidant such as ascorbic acid or sodiumbisulfite; a chelating agent such as etheylenediaminetetraacetic acid; abuffer such as acetate, citrate, or phosphate; and/or an agent for theadjustment of tonicity such as sodium chloride, dextrose, or apolyalcohol such as mannitol or sorbitol. The pH of the aqueous solutionmay be adjusted with acids or bases such as hydrochloric acid or sodiumhydroxide. Oil-based solutions or suspensions may further comprisesesame, peanut, olive oil, or mineral oil.

The compositions may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carried, for example water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets.

For topical (e.g., transdermal or transmucosal) administration,penetrants appropriate to the barrier to be permeated are generallyincluded in the preparation. Pharmaceutical compositions adapted fortopical administration may be formulated as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosolsor oils. In some embodiments, the pharmaceutical composition is appliedas a topical ointment or cream. When formulated in an ointment, theactive ingredient may be employed with either a paraffinic or awater-miscible ointment base. Alternatively, the active ingredient maybe formulated in a cream with an oil-in-water cream base or awater-in-oil base. Pharmaceutical compositions adapted for topicaladministration to the eye include eye drops wherein the activeingredient is dissolved or suspended in a suitable carrier, especiallyan aqueous solvent. Pharmaceutical compositions adapted for topicaladministration in the mouth include lozenges, pastilles and mouthwashes. Transmucosal administration may be accomplished through the useof nasal sprays, aerosol sprays, tablets, or suppositories, andtransdermal administration may be via ointments, salves, gels, patches,or creams as generally known in the art.

In certain embodiments, a composition comprising a compound thatincreases GRIN2B expression or activity is encapsulated in a suitablevehicle to either aid in the delivery of the compound to target cells,to increase the stability of the composition, or to minimize potentialtoxicity of the composition. As will be appreciated by a skilledartisan, a variety of vehicles are suitable for delivering a compositionof the present invention. Non-limiting examples of suitable structuredfluid delivery systems may include nanoparticles, liposomes,microemulsions, micelles, dendrimers and other phospholipid-containingsystems. Methods of incorporating compositions into delivery vehiclesare known in the art.

In one alternative embodiment, a liposome delivery vehicle may beutilized. Liposomes, depending upon the embodiment, are suitable fordelivery of a compound that increases GRIN2B expression or activity inview of their structural and chemical properties. Generally speaking,liposomes are spherical vesicles with a phospholipid bilayer membrane.The lipid bilayer of a liposome may fuse with other bilayers (e.g., thecell membrane), thus delivering the contents of the liposome to cells.In this manner, a compound that increases GRIN2B expression or activitymay be selectively delivered to a cell by encapsulation in a liposomethat fuses with the targeted cell's membrane.

Liposomes may be comprised of a variety of different types ofphosolipids having varying hydrocarbon chain lengths. Phospholipidsgenerally comprise two fatty acids linked through glycerol phosphate toone of a variety of polar groups. Suitable phospholids includephosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol(PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG),phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The fattyacid chains comprising the phospholipids may range from about 6 to about26 carbon atoms in length, and the lipid chains may be saturated orunsaturated. Suitable fatty acid chains include (common name presentedin parentheses) n-dodecanoate (laurate), n-tretradecanoate (myristate),n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate(arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate),cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate),cis,cis-9,12-octadecandienoate (linoleate), all cis-9, 12,15-octadecatrienoate (linolenate), and allcis-5,8,11,14-eicosatetraenoate (arachidonate). The two fatty acidchains of a phospholipid may be identical or different. Acceptablephospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS,distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl,oleoyl PS, palmitoyl, linolenyl PS, and the like.

The phospholipids may come from any natural source, and, as such, maycomprise a mixture of phospholipids. For example, egg yolk is rich inPC, PG, and PE, soy beans contains PC, PE, PI, and PA, and animal brainor spinal cord is enriched in PS. Phospholipids may come from syntheticsources too. Mixtures of phospholipids having a varied ratio ofindividual phospholipids may be used. Mixtures of differentphospholipids may result in liposome compositions having advantageousactivity or stability of activity properties. The above mentionedphospholipids may be mixed, in optimal ratios with cationic lipids, suchas N-(1-(2,3-dioleolyoxy)propyl)-N,N,N-trimethyl ammonium chloride,1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,3,3′-deheptyloxacarbocyanine iodide,1,1′-dedodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate,1,1′-dioleyl-3,3,3′,3′-tetramethylindo carbocyanine methanesulfonate,N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or1,1,-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchloarate.

Liposomes may optionally comprise sphingolipids, in which spingosine isthe structural counterpart of glycerol and one of the one fatty acids ofa phosphoglyceride, or cholesterol, a major component of animal cellmembranes. Liposomes may optionally contain pegylated lipids, which arelipids covalently linked to polymers of polyethylene glycol (PEG). PEGsmay range in size from about 500 to about 10,000 daltons.

Liposomes may further comprise a suitable solvent. The solvent may be anorganic solvent or an inorganic solvent. Suitable solvents include, butare not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone,N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide,tetrahydrofuran, or combinations thereof.

Liposomes carrying a compound that increases GRIN2B expression oractivity (i.e., having at least one methionine compound) may be preparedby any known method of preparing liposomes for drug delivery, such as,for example, detailed in U.S. Pat. Nos. 4,241,046, 4,394,448, 4,529,561,4,755,388, 4,828,837, 4,925,661, 4,954,345, 4,957,735, 5,043,164,5,064,655, 5,077,211 and 5,264,618, the disclosures of which are herebyincorporated by reference in their entirety. For example, liposomes maybe prepared by sonicating lipids in an aqueous solution, solventinjection, lipid hydration, reverse evaporation, or freeze drying byrepeated freezing and thawing. In a preferred embodiment the liposomesare formed by sonication. The liposomes may be multilamellar, which havemany layers like an onion, or unilamellar. The liposomes may be large orsmall. Continued high-shear sonication tends to form smaller unilamellarlipsomes.

As would be apparent to one of ordinary skill, all of the parametersthat govern liposome formation may be varied. These parameters include,but are not limited to, temperature, pH, concentration of methioninecompound, concentration and composition of lipid, concentration ofmultivalent cations, rate of mixing, presence of and concentration ofsolvent.

In another embodiment, a composition of the invention may be deliveredto a cell as a microemulsion. Microemulsions are generally clear,thermodynamically stable solutions comprising an aqueous solution, asurfactant, and “oil.” The “oil” in this case, is the supercriticalfluid phase. The surfactant rests at the oil-water interface. Any of avariety of surfactants are suitable for use in microemulsionformulations including those described herein or otherwise known in theart. The aqueous microdomains suitable for use in the inventiongenerally will have characteristic structural dimensions from about 5 nmto about 100 nm. Aggregates of this size are poor scatterers of visiblelight and hence, these solutions are optically clear. As will beappreciated by a skilled artisan, microemulsions can and will have amultitude of different microscopic structures including sphere, rod, ordisc shaped aggregates. In one embodiment, the structure may bemicelles, which are the simplest microemulsion structures that aregenerally spherical or cylindrical objects. Micelles are like drops ofoil in water, and reverse micelles are like drops of water in oil. In analternative embodiment, the microemulsion structure is the lamellae. Itcomprises consecutive layers of water and oil separated by layers ofsurfactant. The “oil” of microemulsions optimally comprisesphospholipids. Any of the phospholipids detailed above for liposomes aresuitable for embodiments directed to microemulsions. A compound thatincreases GRIN2B expression or activity may be encapsulated in amicroemulsion by any method generally known in the art.

In yet another embodiment, a compound that increases GRIN2B expressionor activity may be delivered in a dendritic macromolecule, or adendrimer. Generally speaking, a dendrimer is a branched tree-likemolecule, in which each branch is an interlinked chain of molecules thatdivides into two new branches (molecules) after a certain length. Thisbranching continues until the branches (molecules) become so denselypacked that the canopy forms a globe. Generally, the properties ofdendrimers are determined by the functional groups at their surface. Forexample, hydrophilic end groups, such as carboxyl groups, wouldtypically make a water-soluble dendrimer. Alternatively, phospholipidsmay be incorporated in the surface of a dendrimer to facilitateabsorption across the skin. Any of the phospholipids detailed for use inliposome embodiments are suitable for use in dendrimer embodiments. Anymethod generally known in the art may be utilized to make dendrimers andto encapsulate compositions of the invention therein. For example,dendrimers may be produced by an iterative sequence of reaction steps,in which each additional iteration leads to a higher order dendrimer.Consequently, they have a regular, highly branched 3D structure, withnearly uniform size and shape. Furthermore, the final size of adendrimer is typically controlled by the number of iterative steps usedduring synthesis. A variety of dendrimer sizes are suitable for use inthe invention. Generally, the size of dendrimers may range from about 1nm to about 100 nm.

(b) Administration

In certain aspects, a therapeutically effective amount of a compositionof the invention may be administered to a subject. Administration isperformed using standard effective techniques, including peripherally(i.e. not by administration into the central nervous system) or locallyto the central nervous system. Peripheral administration includes but isnot limited to intravenous, intraperitoneal, subcutaneous, pulmonary,transdermal, intramuscular, intranasal, buccal, sublingual, orsuppository administration. Local administration, including directlyinto the central nervous system (CNS) includes but is not limited to viaa lumbar, intraventricular or intraparenchymal catheter or using asurgically implanted controlled release formulation.

Pharmaceutical compositions for effective administration aredeliberately designed to be appropriate for the selected mode ofadministration, and pharmaceutically acceptable excipients such ascompatible dispersing agents, buffers, surfactants, preservatives,solubilizing agents, isotonicity agents, stabilizing agents and the likeare used as appropriate. Remington's Pharmaceutical Sciences, MackPublishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest edition,incorporated herein by reference in its entirety, provides a compendiumof formulation techniques as are generally known to practitioners.

For therapeutic applications, a therapeutically effective amount of acomposition of the invention is administered to a subject. A“therapeutically effective amount” is an amount of the therapeuticcomposition sufficient to produce a measurable response (e.g., enhancedmemory performance, reduced memory loss, an improvement in symptomsassociated with cognitive decline, or an improvement in symptomsassociated with dementia). Actual dosage levels of active ingredients ina therapeutic composition of the invention can be varied so as toadminister an amount of the active compound(s) that is effective toachieve the desired therapeutic response for a particular subject. Theselected dosage level will depend upon a variety of factors includingthe activity of the therapeutic composition, formulation, the route ofadministration, combination with other drugs or treatments, age, thecognitive decline, the dementia, the symptoms, and the physicalcondition and prior medical history of the subject being treated. Insome embodiments, a minimal dose is administered, and dose is escalatedin the absence of dose-limiting toxicity. Determination and adjustmentof a therapeutically effective dose, as well as evaluation of when andhow to make such adjustments, are known to those of ordinary skill inthe art of medicine.

The frequency of dosing may be daily or once, twice, three times or moreper week or per month, as needed as to effectively treat the symptoms.The timing of administration of the treatment relative to the diseaseitself and duration of treatment will be determined by the circumstancessurrounding the case. Treatment could begin immediately, such as at thetime of determining the presence or absence of the A allele. Treatmentcould begin at a later time after determining the presence or absence ofthe A allele. Duration of treatment could range from a single doseadministered on a one-time basis to a life-long course of therapeutictreatments.

Typical dosage levels can be determined and optimized using standardclinical techniques and will be dependent on the mode of administration.

In addition to the composition comprising a compound that increasesGRIN2B activity or expression, standard treatments for memory decline,cognitive decline and/or dementia may be administered. Non-limitingexamples of treatment for memory decline, cognitive decline and/ordementia include drugs to boost neurotransmitter levels (e.g., Aricept,Razadyne and Exelon), drugs to regulated the activity of theneurotransmitter glutamate (e.g., memantine), occupational therapy,environmental approaches (e.g., reduce clutter and/or noise), donepezil,vitamin E, diet and exercise, and cognitive rehabilitation.

EXAMPLES

The following examples are included to demonstrate various embodimentsof the present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Introduction to the Examples

The earliest and the most severe memory loss in old age and dementiaoccurs in recent memory and working memory. Compared to young adults,older adults exhibit deficits in working memory, an online memorymechanism that allows manipulation of a current target image amongstdistracting stimuli. Remote memory or long-term is better retained.Evidence from neuropsychological and neuroimaging studies of visualworking memory indicates that working memory relies on activation of theventral temporal cortex, and top-down feedback from the prefrontal andmedial temporal cortex, and the hippocampus. It has been observed thatthere are significant individual differences in cognitive aging amonghealthy cognitively intact older adults due to genetic, learning andenvironmental factors. Since genetic factors influence working memoryperformance in the aging brain and the exact underlying mechanisms arenot well understood, the present study investigates the variation of afunctional promoter polymorphism critical to molecular foundation oflearning and memory in individuals' brain processing speed and corticalresponses during a working memory task.

The N-methyl-D-aspartate (NMDA) receptors are a class of ionotropicglutamate receptors known to play important roles in cellular andmolecular mechanisms associated with efficiency of synaptic transmissionduring memory. The present study tested the hypothesis that a singlenucleotide polymorphism (SNP) within the promoter of the GRIN2B gene(r53764030 G>A) was functional and that function-guided genotypes couldbe used to predict brain responses and memory performance (reactiontimes and accuracy) during working memory. The influence of sequencepolymorphisms potentially affecting expression of NMDAR subunits onbrain function was determined.

E-twenty-six (ETS) transcription factors are a large class ofevolutionarily related, DNA binding proteins. They are characterized bya DNA binding domain that targets a core DNA sequence having a coresequence of 5′-GGA(A/T)-3′. In humans, there are 28 known members of theETS family, which has been subdivided into 12 different subgroups. Inaddition to the core sequence, it is thought that flanking sequencescontribute to binding specificity of ETS family proteins. Using a genomewide approach, evidence for DNA sequence selectivity of among ETSprotein family members has been determined. Other genome wide studieshave combined chromatin immunoprecipitation with microarrays ormassively parallel sequence analysis.

A potential ETS transcription factor binding site created by a G>Asingle nucleotide polymorphism (SNP r53764030) that is 310 bps upstreamof the transcription start site for the human GRIN2B gene promoter wasidentified (FIG. 1). Here, an association of a gain-of-function SNP inthe GRIN2B gene and memory performance in a normally aged population ofadults is described. A function-guided approach was used to analyzegenotype groups based on the observation that the A allele of r53764030creates a possible ETS core binding site. In vitro and in vivo datasupporting a functional role for this sequence variant is provided.

Brain processing speed reduces over aging brain. It was predicted thatvariations in GRIN2B may partially count for individual differences inreaction times during active short-term memory retrieval. In addition,neuroimaging studies have shown increased activation within bilateraldorsal lateral prefontal cortices (DPFC) during a working memory task.The cognitive processes become less modular with age, to providefunctional compensation in the elderly. It is expected that the GRIN2Bgene altered binding of a transcription factor influences corticalresponses during memory retrieval.

Example 1 The A Allele of rs3764030 Rreates an In Vitro DNA Binding Site

The hypothesis that a SNP (rs r53764030, G>A) within the promoter of thehuman GRIN2B gene alters binding of E-twenty six (ETS) familytranscription factors was tested. An in vitro DNA binding assay wasdeveloped using purified Elk-1, an ETS protein family member that isexpressed in brain and DNA targets having either a consensus ETS corebinding site (GGAA/T) or DNA targets based on the presence of the ETSbinding site from the human GRIN2B gene promoter (GGAA, A allele) orabsence of the ETS core binding site (GGGA, G allele). The results inFIG. 2 show that the A allele DNA probe bound recombinant Elk-1 to agreater extent than the G allele DNA probe. When the same gel wasstained for protein, it was readily apparent that the A allele DNA probehad more protein associated with it than the G allele DNA probe (FIG.2).

Example 2 The A Allele of rs3764030 has Greater Basal GRIN2B ReporterGene Activity Than the G Allele in Reporter Gene Assays.

A reporter gene experiment was performed to determine if the A allele ofr53764030 was functional under activity-dependent conditions.Neuron-like murine neuroblastoma N2A cells were transfected with each ofthe Elk-1 binding site variants and subsequently treated for four hourswith increasing concentrations of NMDA. Six hours later, luciferaseactivity was measured. A dose-dependent increase in luciferase activityfrom 2.4-11.5-fold was seen for the A allele (FIG. 3). N2A cellstransfected with the G allele luciferase reporter plasmid did notrespond to NMDA treatment. These data support the idea that the A alleleproduces a gain-of-function phenotype.

Example 3 Association of rs3764030 Genotype With Learning Performance

Using the functional data from and in vivo reporter gene experiments andin vitro DNA binding experiments, r53764030 high expression genotypes(A/A, A/G or A allele carriers) and low expression (G/G) genotypes werefunctionally grouped. This function-based genotype stratification wasused to test for association with behavioral performance during memorytests in the study population. Mean reaction time (±SD) was notsignificantly different between A allele carriers and the G/G genotypegroup (583.9 msec±81.09 vs 583.2 msec±53.9; P=0.490 , by two-tailedtest). However, when mean reaction time was analyzed based on age byanalysis of covariance (ANCOVA), genotype group differences becameapparent. Performance as mean reaction time versus age is shown in FIG.4A, for G/G carriers, and FIG. 4B, for A allele carriers (A/G+A/Agenotypes). A significant difference in the slopes of the regressionlines was observed (−3.994±SE 3.460 for A allele carriers vs 5.848±SE2.281 for G/G; P=0.026).

In another measure, the presence of the A allele was not associated withdifferences in learning rate within the study population (85.96±65.35for GG; 65.35±40.40 for A allele carriers; P=0.165). When analyzed byANCOVA based on age, individuals carrying the A allele had slightlyreduced performance with increased age (Slope=−0.217±SE 1.823) while thelearning rate among G/G individuals decreased more rapidly withincreased age (Slope=−2.276±SE 1.641) However, the difference in theslopes of the two regression lines did not reach significance(P=0.409)(data not shown).

To further validate the results shown in FIGS. 4A and 4B, a secondexperiment was conducted with a second, separate group of cognitivelynormal aging subjects. As shown in FIG. 5A, GG carriers had a positivecorrelation between age and reaction times of repeated memory targetmatch. In contrast, as shown in FIG. 5B, A allele carriers (AA and AGgenotypes) did not show such trend; the A allele carriers reactionstimes decreased slightly with age. The slopes of the regression lineswere significantly different. For A allele carriers as in FIG. 5B, theslope is −3.58±SE 0.21, versus GG genotype as in FIG. 5A having a slopeof 13.1±SE 0.20, t(8)=58.5, p<0.001). Although the number of individualstested is small, the differences in performance stratified by thepresence of or absence of the A allele are statistically significant.The results are consistent with those presented in the experiment asshown in FIGS. 4A and 4B above, and is a critical validation step forpredicting performance of an aging subject.

Example 4 Brain Activation Underlying Working Memory and ItsRelationship With GRIN2B

Both groups utilized some common cortical areas: frontal (BA areas 6, 9,10, & 8) and visual cortices, which include fusiform,parahippocampus/hippocampus, middle temporal, precuneus, cingulate, andoccipital regions (p<0.001). During working memory task, both groupsresponded equally in BOLD responses to matches (new or studied targetobjects). However, they differed in fMRI responses to objects that weremismatched to the target objects held in working memory. Specifically,GG group showed significantly stronger fMRI responses in left inferiortemporal (fusiform, L BA area 37; (1.36% signal change) responses to newnonmatch objects (p<0.01 corrected) than that of AG/AA group (1.08%change). The GG group's medial frontal region (BA6, 32) also respondedstronger to studied nonmatches than that of AG/AA group (GG=1.48% vs.AG/AA=1.08%; p<0.01).

The mid-frontal regions (L BA 46/9) showed the same pattern, with the GGgroup (averaged 1.30% signal change) having enhanced fMRI responsesversus the AG/AA group (1.15%, p<0.05).

Discussion for the Examples

Luciferase assays indicated that the A allele responded to NMDA receptoractivation in a dose-dependent manner and that reporter gene activityunder these conditions was significantly higher (2-fold) than cellscarrying the same reporter gene but with the G allele. Results from gelshift experiments indicated that the A allele could create a functionalbinding site for at least one ETS domain transcription factor.Behavioral results showed that presence of the A allele was notassociated with differences in either memory accuracy (97% for GG; 96%for AG/AA) or reaction times (584ms and 589 ms). However, A allelecarriers showed decreased reaction time with age (meaning better memoryperformance) compared with the GG genotype (p=0.026 based on differencein slope of the regression line between GG and AG=AA groups) in a groupof cognitively normal aged adults. This finding was replicated in asecond cohort of cognitively normal subjects. A allele carriersperformed better compared with GG genotype individuals (p<0.001) Brainimaging results revealed that alterations of brain responses in ventraltemporal, and in prefrontal cortices are associated with the changes insubunit of NMDA receptors, which confirms level of facilitation inmemory functions.

In this study, a biological effect of the GRIN2B promoter SNP, r53764030G>A, was determined, showing that the A allele in transfected N2a cells,responded to NMDA agonism (NMDA receptor activation) in a dose-dependentmanner relative to the common G allele. In addition, it was shown thatthe A allele was capable of binding the ETS transcription factor, Elk-1,in vitro. These observations were the mechanistic basis for providingthe support for a gain-of-function gene variant and for combininggenotype groups (A allele carriers vs non-carriers) for subsequentgenetic association studies. The cohort recruited in this study wascomposed of cognitively normal older adults. Better performance inreaction time to a learning test in A allele carriers was determined.

This study is the first report of a SNP that affects GRN2B mRNA levels.The knowledge of a functional polymorphism that may increase levels ofGRIN2B mRNA and possibly GluN2B subunits suggests a mechanism forprotecting the brain from age-related cognitive decline.

The results from the gel shift experiment indicated that the A allelecreated a functional binding site for at least one ETS domaintranscription factor. The ETS domain transcription factor family has 27known members that are expressed by different human cells. The initialfocus was on Elk-1 because its expression profile in different tissuesincludes the brain.

Conclusions. These results support the idea that presence of the Aallele of r53764030 positively influenced reaction time. Alternatively,individuals with the G allele, while having slower responses withincreasing age, may have had a stronger capability to suppressdistractors. Either interpretation is consistent with the idea thatchanges in subunit stoichiometry of NMDA receptors confer distinctfunctional properties of memory functions. The grouping of Aallele-genotypes for association analyses was justified based onreporter gene assays and gain-of-function ETS transcription factorbinding to DNA. Future longitudinal follow-up of the subjects will bringinsights of whether the gene variation might be a potential indicatorfor cognitive reserve or risk-factor for old-age dementia.

Methods for the Examples

Participants: Twenty-eight right-handed older adults (ages 65-86) wererecruited as a cohort to participate in the experiment. Individuals werecognitively normal with no diagnosis of dementia, substance abuse, majorpsychiatric illness, or other illnesses or conditions affecting thecentral nervous system, such as meningitis or traumatic brain injury.Written consent forms were obtained from each of the participants.

Genotyping: Subjects were genotyped for the r53764030 G>A GRIN2Bpromoter SNP using a 5′ exonuclase assay. Genotypes were independentlyconfirmed using direct sequence analysis with no discrepancies. Genotypefrequencies met Hardy-Weinberg expectations in the study population. Forassociation analyses, subjects were grouped based on presence of Aallele (GG genotype versus GA and AA genotypes). Both GG (n=15) andAG/AA (n=13) groups were well matched in age (mean age 75.5 for GG, 74.5for AG/AA), gender (9 female and 8 female respectively). Both genotypegroups had high cognitive functioning with comparable scores on theMini-Mental State Exam (mean=29).

Experimental paradigm: Study Phase. Participants were initiallyinstructed to study and memorize 60 line drawings individually displayedin a computer task, cycling through each by pressing the spacebar tomove onto the next stimulus. This phase lasted approximately 10 minutesper subject. After these two study phases, each participant was asked tocomplete a recognition task during which they identified the picturespresented as “memorized” or “not memorized” by pressing thecorresponding keyboard button (placement of the keys wascounterbalanced). Participants subsequently performed a recognition testthat included 60 studied and 60 new objects (mean accuracy=98.2%).

Stimuli. Stimuli consisted of 120 black and white line-drawings ofcommon objects developed by Snodgrass & Vanderwart (1980). Pictures werepresented within a rectangular area of 8.3 cm by 5.8 cm and displayed infront of a black background. The visual display and responses werecontrolled by E-Prime presentation software (Psychology Software Tools,Pittsburgh, Pa.). The computer screen was approximately 65 cm fromparticipants with a visual angle of about 7 degrees. The targetpictures, at the beginning of each trials, were demarcated by a 6.5 mmgreen border as indication of the item to be held in mind as

Of the 120 pictures, 40 were used as studied distracters, while theother 80were new objects that had not been previously studied (40 newdistracters; 40 new targets). Test objects were classified into one ofthree groups: (a) studied targets, (b) studied distracters, or (c) newdistracters. All target objects were new but distracter objects includedboth studied and new objects.

Test Phase. Participants then performed the working memory task using abutton held in each hand. Assignment of hands was counterbalanced acrosssubjects. For each working memory trial, participants were shown asample object to hold in mind and were asked to indicate whether thefollowing nine test objects were the same or different from the target.Participants then performed a Delayed Match to Sample (DMS) taskconsisted of 40 trials separated into 4 blocks of 10 trials each. Eachtrial began with the presentation of a sample target object for 2000msec and was distinguished by a green border. The sample target objectwas followed (ISI=700±100 msec) by 10 successive test objects with astimulus duration of 2000 msec (ISI=500±200 msec). Each trial lasted27.5 seconds.

The test portion of each trial contained a pseudo-random presentation oftarget and distracter objects where the target object, a studieddistracter, and a new distracter were presented three times each,resulting in nine of the ten test items in a trial. One additional‘filler’ object was included in each trial to reduce the potential forsubject expectancy and served either as a 4^(th) target (16.7% oftrials), 4^(th) studied distracter (16.7% of trials), 4^(th) newdistracter (16.7% of trials), or a new distracter never previously shown(50% of trials). None of the objects, whether serving as a target ordistracter, were used in any subsequent trials. Across trials, stimulifrom the three experimental conditions were equally distributed acrossall 10 serial positions. FIG. 1 illustrates the distribution of objectsin relation to repetition across the 10 serial test object positions.

Participants were told to hold the sample target object in mind andindicate whether the following 10 test objects were the same ordifferent from the sample target. Assignment of hands to indicate atarget versus distracter object was counterbalanced across subjects.Subjects were also instructed to forget the previous sample targetobject only when a new sample target object appeared.

Reaction times and accuracy of behavioral responses were recorded asmsecs and number of correct responses.

Behavioral data analysis: Behavioral data, i.e. accuracy and reactiontimes, were calculated to each of the memory retrieval, new or studiedmemory matching target, new or studied nonmtach distractors. Please seeCaggiano, Jiang, Parasurman, 2006 for detailed description.

Event-related functional MRI Acquisition and Analysis: The healthynormal older adults were scanned inside of a 3 Tesla Siemens Trio MRIscanner at the University of Kentucky. High-resolution whole brainstructural MRI was obtained for each subject. Twenty-two slices wholebrain T2* weighted functional images were obtained every 2.5 seconds foreach of the eight series. [T2*-weighted EPI (64×64 matrix, 2.5 sec RT,whole brain, 3.6 mm cubic voxel size]. Images were realigned for headmotion correction and fMRI Image volumes were reconstructed using AFNIsoftware (Cox, 1996). Motion was corrected and the slice timingdifferences were adjusted and intensity normalized to allow for thecalculation of activation as a percentage of signal change. Generallinear models were applied for the multiple regression analysis. Themultiple regression models contained orthogonal contrasts of interestand additional regressors of no importance to obtain changes in meanfMRI signals.

Molecular and cellular characterization of a functional GRIN2B SNP:Luciferase vector construction. The r53764030 SNP within the 1.7kb 5′flanking region which covered the GRIN2B gene noncoding exon, forexample exon 1, and 1530 bps upstream of the human GRIN2B genetranscription start site was PCR amplified and cloned into pGL 4.10 (toproduce Firefly luciferase, Promega, US) plasmid for luciferase reportergene assay. The four plasmid constructs were each designed according toa combination of alleles (either A or G) and the DNA strand transcribed,which included: positive orientation with allele A (A+plasmid), positiveorientation with allele G (G+plasmid), negative orientation with alleleA (A−plasmid) and negative orientation with allele G (G−plasmid). Theorientations and alleles in the constructs were confirmed by directsequencing in ABI 310 genetic sequencer (Applied Biosystems, US).

Cell Culture, Transfection, and Luciferase assay. Murine N2a cellsproduce functional NMDA receptors (Mantuano et al 2013; Van der Valk andVijverberg, 1990). The cells were maintained in RPMI medium supplementedwith 5% fetal bovine serum until approximately 80% confluent. Prior totransfection the cultures were transferred into 96 well cell cultureplates (Costar #3904, US) 24-48 hours with serum free RPMI medium 1640.The four plasmid constructs were co-transfected with pGL4.75 plasmid (toproduce Renilla luciferase as control) for dual-luciferase reporter geneassay (Promega, US) using Lipofextamine-LTX reagent (Life science, US).After 24 hrs of transfection, the cells were activated 4-6 hours with 0,30, 50, 70 and 90 uM NMDA for transcription factors binding and thenreplaced with completed media (with 10% FBS) to cultured additional40-44 hrs at 37° C. in 5% CO2. Each NMDA concentration was replicatedthree times for statistical power. The luciferase activities were thenmeasured in Synergy 4 plate reader (Biotek, US).

Murine Neuro-2a neuroblastoma cells were maintained in RPMI medium 1640with 10% Fetal Bovine Serum (complete medium) at 37° C. in 5% CO2. Cellswere plated in 96 well cell culture plates (1×10⁶ cells) with lamininand Poly-L-Ornithine) 24 hrs before neural differentiation. Fordifferentiation, cells were incubated in the presence of 1 μM retinoicacid for 24 hrs in serum-free RPMI medium. The N2a cells weredifferentiated on 96 well cell culture plate (Costar #3904, US) 24-48hours with serum free RPMI medium 1640 before transfection. The 4plasmid constructs were co-transfected with pGL4.75 plasmid (to produceRenilla luciferase as control) for dual-luciferase reporter gene assay(Promega, US) using Lipofextamine-LTX reagent (Life science, US). After24 hours of transfection, the cells were activated 4-6 hours with 0, 30,50, 70 and 90 uM NMDA for transcription factors binding and thenreplaced with completed media (with 10% FBS) to cultured additional40-44 hours at 37° C. in 5% CO2. Each NMDA concentration was replicatedthree times for statistical power. The luciferase activities were thenmeasured in Synergy 4 plate reader (Biotek US).

Electrophoretic Mobility Shift Assay (EMSA). To monitor the interactionbetween the ETS domain transcription factor, Elk-1, and its DNArecognition sequence in vitro, we used EMSA. The DNA sequences forpositive control containing a GGAT consensus core binding sequence were:Forward: 5′ ACGCTAACCGGATATAACGCTA 3′ (SEQ ID NO:2) and Reverse: 5′TAGCGTTATATCCGGTTAGCGT 3′ (SEQ ID NO:3). The negative control regionwas: Forward: 5′ ACGCTAAACAGTGTCAACGCTA 3′ (SEQ ID NO:4) and Reverse: 5′TAGCGTTGACACTGTTTAGCGT 3′ (SEQ ID NO:5), as described in Wei et al(2010). The forward and reverse sequence for GRIN2B A allele whichcreated the ETS binding site were: 5′ CATCTCCGGGGAACACGCGAA 3′ (SEQ IDNO:6)and 5′ TTCGCGTGTTCCCCGGAGATG 3′ (SEQ ID NO:7), respectively. Theforward and reverse sequences for GRIN2B G allele were 5′CATCTCCGGGGGACACGCGAA 3′ (SEQ ID NO:8) and 5′ TTCGCGTGTCCCCCGGAGATG 3′(SEQ ID NO:9).

The complementary pairs of DNA oligonucleotides were first annealed inBiorad C1000 thermocycler with 1° C./1 min decreasing from 95° C. to 20°C. The annealing buffer composition was 10mM Tris-HCl pH 8.0, 1mM EDTAand 50mM NaCl. The annealed dsDNA was then incubated with 1 μgrecombinant human Elk-1 protein (Sigma Aldrich, US) on ice for 1 hour inEMSA buffer, then incubated with the Elk-1 antibody (SC-355)(Santa CruzTechnology, US) 20 minutes in room temperature. The compositions of EMSAbuffer was 50 mM HEPES pH7.5, 10 mM MgCl₂, 5% glycerol, 1 mM DTT, 0.3%BSA and 1mM EDTA. The experiment was conducted in 6% retardation gel(Life Technology, US), running 95 volt for 80 minutes. The gel was thenstained with the EMSA SYBR Green and SYPRO Ruby kit (E-33075)(Lifetechnology, US) according to the instruction manual. The stained 6%retardation gel was scanned in G:BOX (Syngene, US) for imaging.

This work was partially funded by grants P30 AG028383 and K01 AG000986awarded by NIH, the disclosure of which are hereby incorporated byreference in their entirety.

What is claimed is:
 1. A method to delay the onset of cognitive declinein a subject, the method comprising administering a compositionincluding a compound that increases GRIN2B activity or expression. 2.The method of claim 1, wherein the cognitive decline is due to dementia.3. A method for identifying a candidate individual for administering acomposition comprising a compound that increases GRIN2B activity orexpression, the method comprising: a) analyzing an MRNA transcriptincluding a GRIN2B nucleic acid sequence for the presence of an A allelein a biological sample obtained from the subject; b) classifying thebiological sample as (i) having the A allele if GGAA is detected 310base pairs upstream of the transcription start site for the GRIN2Bnucleic acid sequence or (ii) not having the A allele if GGGA isdetected 310 base pairs upstream of the transcription start site for theGRIN2B nucleic acid sequence; and c) administering the composition tothe biological sample; d) conducting testing to determine response ofthe biological sample to the composition; e) analyzing an MRNAtranscript of an individual including a GRIN2B nucleic acid sequence forthe presence of an A allele in a biological sample obtained from thesubject; f) classifying the individual as (i) having the A allele ifGGAA is detected 310 base pairs upstream of the transcription start sitefor the GRIN2B nucleic acid sequence or (ii) not having the A allele ifGGGA is detected 310 base pairs upstream of the transcription start sitefor the GRIN2B nucleic acid sequence; g) determining whether theindividual is or is not a candidate for administering the compositionbased on the classification of the individual from the presence orabsence of the A allele.
 4. The method of claim 3, wherein the A alleleis detected by sequencing.
 5. The method of claim 3, wherein the Aallele is detected by hybridization of a probe specific to the A allele.6. The method of claim 5, wherein the probe comprises SEQ ID NO:6(CATCTCCGGGGAACACGCGAA).
 7. The method of claim 3, wherein SNP r53764030is detected about 310 base pairs upstream of the transcription startsite for the GRIN2B nucleic acid sequence.
 8. The method of claim 3,wherein the candidate is homozygous for the A allele if only GGAA isdetected.
 9. The method of claim 3, wherein the candidate isheterozygous for the A allele if GGAA and GGGA are detected.